The impressively large absorptive surface area of the intestine is largely contributed by its length as well as the extensive surface amplification provided by millions of fingerlike villus projections. Congenital or acquired pathologies that result in significant loss of this intestinal surface area seriously compromise the ability of the intestine to absorb nutrients and can be life threatening. Work in the past project period has begun to identify some of the cellular and molecular mechanisms that control the formation of villi in utero in the murine model. The period between embryonic day (E) 14.5 and E15.5 (in the mouse) is particularly important since during this time, controlled morphogenic remodeling in both the epithelium and the underlying mesenchyme results in the emergence of villi. The Working Hypothesis underlying these studies is that: Villus development is actively inhibited by Bmp signaling prior to E14.5. At E14.5, epithelial Hh signals initiate villus development by promoting the formation of mesenchymal clusters. Cluster patterning proceeds via a Turing system and vascular coupling to these clusters precedes and is required for villus emergence. Importantly, since the morphological hallmarks of all of these events are also present in the human intestine, it is likely that the majority of the signaling paradigms uncovered in the mouse model will be applicable to the human intestine. This proposal makes use of genetic mouse models as well as a novel intestinal explant culture system to mechanistically dissect the interconnected signaling and patterning events involved in these surface- generating processes. Additionally, a new software tool that can recognize Hh-responsive signaling enhancers in genomic DNA with high efficiency will aid in the recognition of Hh target genes. The Specific Aims are designed to 1) Determine how Bmp signaling controls competence to form villi and establish which tissue (epithelium or mesenchyme) exerts this control; 2) Identify Hh target genes during the formation of mesenchymal clusters; and 3) Determine the relationship between vascular elements, forming clusters and cluster pattern. Through detailed analysis of these linked processes, the goal of these studies is to gain new insight into the formation of the intestinal absorptive surface. The ability to bioengineer organs from their cellular components will require not only that we elucidate the molecular signals that are important for morphogenesis and cell fate determination, but also that we understand the rules that govern the patterning of the functional units that comprise the organ. The unique focus of this investigation on signaling crosstalk in the nascent villus unit (epithelium, mesenchyme, vasculature) will have a major impact on our understanding of how intestinal villi are first formed in the embryo. PUBLIC HEALTH RELEVANCE: The surface of the small intestine is highly convoluted by finger-like projections called villi; this extended surface area is critical for efficient nutrient absorption. Loss of intestinal surface area either by congenital intestinal defects or by pathological or surgical events, can be life threatening. The ability to bioengineer the intestine from its cellular components will require not only that we elucidate the molecular signals that are important for morphogenesis and cell fate determination, but also that we understand the rules that govern the patterning of the functional units that comprise the organ. Currently, little is known about how villi are generated in utero. Through the study of mouse models with perturbed villus formation and through the analysis of a novel intestinal explant culture system, we propose to dissect the molecular processes responsible for villus emergence. The unique focus of this investigation on signaling crosstalk in the nascent villus unit (epithelium, mesenchyme, vasculature) will have a major impact on our understanding of how intestinal villi are first formed in the embryo.